Molecular imaging can be broadly defined as the characterization and measurement of biological processes at the cellular and molecular level in mammals and human patients. In contradistinction to “classical” diagnostic imaging, for example, magnetic resonance (MR), computed tomography (CT), and ultrasound (US) imaging, molecular imaging analyses molecular abnormalities that are the basis of disease, rather than imaging the end-effects of these molecular alterations. Specific imaging of molecular targets allows earlier detection and characterization of disease, as well as earlier and direct molecular assessment of treatment efficacy. Molecular imaging can theoretically be performed with different imaging technologies, up to now preferably with nuclear imaging technologies, e.g., PET and SPECT imaging) which have high sensitivity of probe detection. The IV administered imaging probes typically recognize a given target. Alternatively, some probes detectable by MR imaging have been developed (Moats et al., Angewandte Chemie Int. Ed., 36:726-731, 1997; Weissleder et al., Nat. Med., 6:351-5, 2000), although their detection threshold is generally in the micromolar instead of the pico/femptomolar range of isotope probes.
An alternative method is to use fluorescent probes for target recognition. For example, enzyme activatable fluorochrome probes are described in Weissleder et al., U.S. Pat. No. 6,083,486, and fluorescent molecular beacons that become fluorescent after DNA hybridization are described in Tyagi et al., Nat. Biotechnol., 16:49-53, 1998. Fluorescence activatable probes have been used in tissue culture and histologic sections and detected using fluorescence microscopy. When administered in vivo, fluorescence activatable probes have been detected by surface-weighted reflectance imaging (Weissleder et al., Nat. Biotechnol., 17:375-8, 1999); Mahmood et al., Radiology, 213:866-70, 1999. However, imaging in deep tissues (>5 mm from the surface), in absorbing and scattering media such as mammalian tissues, and quantitating fluorescence (and in particular fluorescence activation) has not been described.
To image light interactions in deeper tissues, light in the near infrared (near-IR or NIR) instead of the visible spectrum is preferred. Imaging with near infrared (near-IR or NIR) light has been in the frontier of research for resolving and quantifying tissue function. Light offers unique contrast mechanisms that can be based on absorption, e.g., probing of hemoglobin concentration or blood saturation, and/or fluorescence, e.g., probing for weak auto-fluorescence, or exogenously administered fluorescent probes (Neri et al., Nat. Biotech., 15:1271-1275, 1997; Ballou et al., Cancer Immunol. Immunother., 41:257-63,1995; and Weissleder, 1999). In either application, NIR photons undergo significant elastic scattering when traveling through tissue. This results in light “diffusion” in tissue that hinders resolution and impairs the ability to produce diagnostically interpretable images using simple “projection” approaches (transillumination), as in x-ray imaging.
During the last decade, mathematical modeling of light propagation in tissue, combined with technological advancements in photon sources and detection techniques has made possible the application of tomographic principles (Kak and Slaney, “Principles of Computerized Tomographic Imaging,” IEEE Press, New York, 1988, pp. 208-218); Arridge, Inverse Problems, 15:R41 -R93, 1999) for imaging with diffuse light. Diffuse Optical Tomography (DOT) uses multiple projections and deconvolves the scattering effect of tissue. DOT imaging has been used for quantitative, three-dimensional imaging of intrinsic absorption and scattering (see, e.g., Ntziachristos et al., Proc. Natl. Acad. Sci., USA, 97:2767-72, 2000, and also Benaron et al., J. Cerebral Blood Flow Metabol., 20(3):469-77, 2000). These fundamental quantities can be used to derive tissue oxy- and deoxy-hemoglobin concentrations, blood oxygen saturation (Li et al., Appl. Opt., 35:3746-3758, 1996) or hematoma detection in diffuse media.
Although intrinsic-contrast for DOT imaging may be useful in certain situations, e.g., for functional brain activation studies or hematoma detection, these applications do not allow the extraction of highly specific molecular information from living tissues. Fluorochrome concentration has been measured by absorption measurements (Ntziachristos et al., 2000) or by fluroescence measurements in phantoms (Chang et al., IEEE Trans. Med. Imag., 16:68-77, 1997; Sevick-Muraca et al., Photochem. Photobiol., 66:55-64, 1997). However, previously described DOT systems and/or image algorithms have not been useful to obtain three-dimensional quantitation of fluorescence in deep tissues in living mammals.